We have shown that herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) expression is important for HSV latency. Latency was defined by the usual criterion of the isolation of infectious virus during the period of latent infection. For the isolation of the virus it is necessary that (1) the latent infection be established and (2) that infectious virus can be reactivated. In the present proposal we plan to differentiate between the role of HSV TK expression for the establishment of latency and for the reactivation of the latent virus. To perform this detailed investigation of the mechanisms of latency we will perform studies with a temperature sensitive TK- mutant of HSV-1 and a mutant of HSV-1 containing a defined deletion in the TK gene. Studies will also be performed with TK mutants of HSV-2, including a deletion mutant to determine the importance of TK expression in the pathogenesis of infection. In vivo complementation to determine latency established by TK- HSV, rescue of the TK- HSV during latency, and detection of TK- HSV by in situ hybridization will be evaluated. In situ hybridization methodology will also be employed to investigate the presence of HSV specified RNA, including that for TK activity during latency. Finally, methods to inhibit and to chronize HSV reactivation will be utilized to permit study of the reactivation process. These detailed studies of the establishment of HSV latency and of reactivation will provide insights into the mechanisms of these processes.